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Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production.

van der Veen BA, Uitdehaag JC, Penninga D, van Alebeek GJ, Smith LM, Dijkstra BW, Dijkhuizen L
J Mol Biol 296 1027-38 (2000)
Cited: 43 times
EuropePMC logo PMID: 10686101

Abstract

Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 A resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity.

Reviews citing this publication (11)

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  12. Comparative study of the cyclization reactions of three bacterial cyclomaltodextrin glucanotransferases. Terada Y, Sanbe H, Takaha T, Kitahata S, Koizumi K, Okada S. Appl Environ Microbiol 67 1453-1460 (2001)
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  18. Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase. Leemhuis H, Dijkstra BW, Dijkhuizen L. Eur J Biochem 270 155-162 (2003)
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  22. Enhancing the α-Cyclodextrin Specificity of Cyclodextrin Glycosyltransferase from Paenibacillus macerans by Mutagenesis Masking Subsite -7. Wang L, Duan X, Wu J. Appl Environ Microbiol 82 2247-2255 (2016)
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Related citations provided by authors (3)

  1. Engineering of Cyclodextrin Product Specificity and pH Optima of the Thermostable Cyclodextrin Glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1. Wind RD, Uitdehaag JCM, Buitelaar RM, Dijkstra BW, Dijkhuizen L J. Biol. Chem. 273 5771-5779 (1998)
  2. Structure of Cyclodextrin Glycosyltransferase Complexed with a Maltononaose Inhibitor at 2.6 Angstrom Resolution. Implications for Product Specificity. Strokopytov B, Knegtel RM, Penninga D, Rozeboom HJ, Kalk KH, Dijkhuizen L, Dijkstra BW Biochemistry 35 4241-4249 (1996)
  3. Site Directed Mutagenesis in Tyrosine 195 of Cyclodextrin Glycosyltransferase from Bacillus circulans Strain 251 Affect Activity and Product Specificity. Penninga D, Strokopytov B, Rozeboom HJ, Lawson CL, Dijkstra BW, Bergsma J, Dijkhuizen L Biochemistry 34 3368-3376 (1995)

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